Project Summary Activation of the COX-2/mPGES-1/PGE2 signaling axis is a hallmark of many cancers, including colorectal (CRC), leading to the implementation of therapeutic strategies for targeting COX-2 activity. Despite their demonstrated chemopreventive efficacy, long-term treatment with COX inhibitors poses significant health risks as a result of the global suppression of physiological prostanoids. We have shown that targeting the downstream terminal PGE2 synthase, mPGES-1 (Ptges), specifically reduces inducible PGE2 formation and confers protection against colon carcinogenesis in several mouse colon cancer models. In our lipidomic analysis to define the redistribution of the COX products, we found significant metabolic shunting towards PGD2 and thromboxane B2 (TXB2) in the colon tumors of mPGES-1-deficient Apc-mutant mice. These metabolites are primarily produced by mast cells, a cell type that has been frequently detected within the tumor microenvironment. Prostanoids have been shown to directly influence the functional activity of mast cells, and a large body of evidence suggests that mast cell-derived mediators including PGD2 contribute to cancer pathogenesis. These observations serve as a basis for our hypothesis that mPGES-1 targeting causes metabolite shifts in the mast cells, which alters tumor microenvironment towards less growth-promoting. We propose experiments to develop a better understanding of functional roles of mast cells on colon tumor development, and how genetic targeting of mPGES-1 will affect its actions. Aim 1 will extend our pilot lipidomic analysis of colon tumors to confirm the extent of prostanoid shunting and also to identify metabolically distinct prostanoid profiles that are associated with both size and macroscopic features of Apc-driven tumors formed in the presence or absence of mPGES-1. Aim 2 will isolate tumor-associated mast cells from the colon of Apc mice and examine the impact of mPGES-1 blockade on mast cell activation. We will measure the release and production of mediators including histamine, proteases and a panel of prostanoids and cytokines, which directly impact the tumor microenvironment. We will also explore how mast cell responds to PGE2, which is often found in high concentrations within the tumor microenvironment. Aim 3 will generate tumor organoids from Apc mice with or without mPGES-1, and test the direct effects of mast cell-derived mediators (Aim 2) on cancer stem cell homeostasis. Results from these studies will shed light on the roles of mPGES-1 activity in mast cells, and also provide a novel paradigm for the tumor protective mechanism by PGE2 inhibition.